Dissertation > Excellent graduate degree dissertation topics show

The Establishment and Application of Micro-entrapment Cultivation in Isolation of Myxobacteria

Author: GaoXiuZhen
Tutor: LiYueZhong
School: Shandong University
Course: Fermentation Engineering
Keywords: myxobacteria Sorangium cellulosum gel beads purify resources exploitation
CLC: TQ920.1
Type: Master's thesis
Year: 2010
Downloads: 161
Quote: 1
Read: Download Dissertation

Abstract

The myxobacteria show typical social behaviors, such as cell density-dependent growth patterns and growing close together. When the inoculum is below 105 cells/ml, it is difficult for the cells to initiate growth, but once they grow up from a strong inoculum, they will pack closely together. Their characteristic of complex cooperative growth has been a barrier for separating myxobacteria, inhibiting the selection of Sorangium cellulosum mutants during the genetic engineering of the epothilone biosynthetic gene cluster, for example.In this work, we used Sorangium cellulosum as a model. A single S.cellulosum cell embedded into alginate gel micro-beads (diameter of formative beads:1.2±0.5 mm) was able to be triggered to form single-cell colonies when the beads were co-incubated with a high concentration of unpacked S.cellulosum. The beads were dissolved on medium containing a high concentration of phosphate, and the released micro-colonies were further cultured in normal growth medium to obtain purified clones. The developed protocol was efficient at separating mixed cultures of S.cellulosum strains that were isolated from the same soil sample and were highly similar. On the one hand, this technology makes use of the gel matrix to limit the clustering of S.cellulosum cells during growth; on the other hand, the cross-linked structures that are formed permit the penetration of nutrients necessary for growth. With the help of the unpacked S.cellulosum cells outside of the beads, the single cell in the gel bead succeeds at overcoming the cell-density requirement and grows up into a colony. We predict that this method will become standard for obtaining single colonies from cooperatively growing myxobacteria.On the basis of the developed method, we purified a S.cellulosum mutant to obtain single colonies using a S.cellulosum wild-type strain as the helper. Interestingly, we obtained single colonies with the genetic traits of the mutant. HPLC revealed that the colonies we purified had substantial differences in the production of epothilone. Compared to the new method, the traditional operation is time-consuming and inefficient. We have reason to believe that the method we established will accelerate the rate of progress and improve the efficiency of genetic engineering in S.cellulosum. In the past, the ability to form fruiting bodies was used to isolate myxobacteria from the soil. There are many properties of myxobacteria during their growth that limit the utilization of myxobacteria. For instance, it is not easy for a cell to form a colony, the fruiting body is not stable and might even degrade, and it is common for myxobacteria to be contaminated by other bacteria and fungi because of their long generation time. Considering these limitations, we used the established method to isolate myxobacteria with the help of unpacked myxobacteria cells of a different genus of the Cystobacterineae suborder. Using phylogenetic analysis of the 16S rRNA gene (only 448 bp), we discovered that there were new myxobacteria in the gel beads, most of which were different from the standard myxobacteria strains and isolated strains from the same soil sample. Unfortunately, after several attempts to culture the strains from the beads, we found there were only a few strains that could be cultured with stimulation of the helper. Sequencing of the entire 16S rRNA gene suggested that the strains from the gel beads were novel myxobacteria strains. The developed protocol will make it possible to exploit novel uncultured myxobacteria.

Related Dissertations

CLC: > Industrial Technology > Chemical Industry > Other chemical industries > Fermentation industry > General issues > Basic theory
© 2012 www.DissertationTopic.Net Mobile